Peptide compositions

ABSTRACT

The present invention relates to polypeptide compounds that are modulators (e.g., agonists and antagonists) of the melanocortin-4 receptor (MC4R) and pharmaceutical compositions comprising same. The compounds described herein are polypeptide of the following structural Formula (I): 
                         
or a pharmaceutically acceptable salt thereof. Values and preferred values of the variables in structural Formula (I) are described herein.

RELATED APPLICATION

This application is a U.S. National Phase Application under 35 U.S.C. §371 of International Application No. PCT/US2014/028590, filed Mar. 14,2014, which claims the benefit of U.S. Provisional Application No.61/790,469, filed Mar. 15, 2013, the contents of which are incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 2, 2014, isnamed R2054-7002WO_SL.txt and is 30,833 bytes in size.

BACKGROUND OF THE INVENTION

Disorders such as obesity, metabolic syndrome, insulin resistance anddiabetes dramatically add to national healthcare costs and can have asevere impact on the quality of life of afflicted individuals, theirfamilies and caregivers. The incidence of these disorders is increasing,approaching epidemic proportions. Accordingly, a need exists forcompositions and methods of treating these disorders.

SUMMARY OF THE INVENTION

The present invention relates to polypeptide compounds that aremelanocortin-4 receptor (MC4R) modulators and pharmaceuticalcompositions comprising same.

In a particular embodiment the polypeptide compound is an isolatedpolypeptide of the following structural Formula (I):

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R¹ is H, or a C1-C6 acyl;    -   R² is —NR³R⁴, or —OR⁵ wherein R³, R⁴, and R⁵ are each        independently is H or a C1-C6 alkyl;    -   A¹ is an amino acid residue selected from Arg, Lys, Orn, His,        Nle, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu,        or TzAla; or    -   A¹ is a moiety selected from an optionally substituted C1-C12        alkyl, an optionally substituted C6-C18 aryl, an optionally        substituted C5-C18 heteroaryl, an aralkyl wherein the aryl        portion is an optionally substituted C6-C18 aryl, and the alkyl        portion is an optionally substituted C1-C12 alkyl, or a        heteroaralkyl, wherein the heteroaryl portion is an optionally        substituted C5-C18 heteroaryl, and the alkyl portion is an        optionally substituted C1-C12 alkyl;    -   A² and A⁸ is each independently an amino acid residue selected        from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr, wherein A²        and A⁸ are pairwise selected so as to be able to form covalent        bond between their respective side chains;    -   A³ is absent or is an amino acid residue selected from Ala, Tle,        Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar,        Gly, Asn, Aib, or residue Y, wherein Y is an amino acid selected        from amino acids represented by the following structural        formulas

wherein:

-   -   R¹¹ and R¹², each independently, is H, —CH₃, phenyl, or benzyl;    -   R²¹, R²², R²³, and R²⁴, each independently is H, —CH₃, —CF₃,        phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   R³¹, R³², R³³, R³⁴, R⁴¹, R⁴², and R⁴³, each independently is H,        —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   A⁴ is absent or is an amino acid residue selected from Atc, Ala,        QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, an optionally        substituted His, Trp, Tyr, Lys, Arg, sChp, or residue X, where        the X is an amino acid selected from amino acids represented by        the following structural formulas:

wherein:

-   -   R⁵¹ and R⁵², each independently, is H, —CH₃, phenyl, or benzyl;    -   R⁶¹, R⁶², R⁶³, and R⁶⁴, each independently is H, —CH₃, —CF₃,        phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   R⁷¹, R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², and R⁸³, each independently is H,        —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   A⁵ is an optionally substituted Phe, optionally substituted        1-Nal, or an optionally substituted 2-Nal;    -   A⁶ is Arg; and    -   A⁷ is Trp,

wherein any amino acid residue is either in L- or in D-configuration,

provided that:

-   -   1) A³ and A⁴ are not both absent;    -   2) when A⁴ is an amino acid, A³ is not Aib or Gly; and    -   3) when A⁴ is His and A⁵ is a D-Phe or 2-Nal, A³ is not a        D-amino acid or L-Ala;    -   4) when A² and A⁸ each is selected from Cys, hCys or Pen, then:        -   (a) when A⁴ is absent, then A³ is not L-His;        -   (b) when A³ is absent, then A⁴ is not L-His; and        -   (c) when A⁴ is His, then A³ is not Glu, Leu, or Lys.

The present invention also relates to a method of treating a disorderresponsive to the modulation of MC4R in a subject in need of treatment.The method comprises administering to the subject an effective amount ofan MC4R modulator described herein. In a particular embodiment, thedisorder responsive to modulation of the MC4R includes type 1 diabetes,type 2 diabetes, obesity, insulin resistance, metabolic syndrome, maleerectile dysfunction, female sexual disorder, non-alcoholic fatty liverdisease, non-alcoholic steatohepatitis, disorders of substance abuse,including alcoholism feeding disorders, cachexia, inflammation andanxiety.

In certain embodiments, the compounds and compositions of the presentinvention possess higher selectivity and potency for the MC4R andmelanocortin-3 receptor (MC3R) when compared to melanocortin-1 receptor(MC1R). The compounds and compositions of the present invention canreduce or eliminate such undesirable side effects as increase in bloodpressure effects, increase in heart rate, undesired effects on sexualarousal, and increase in skin pigmentation.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Glossary

The nomenclature used to define the peptides is that typically used inthe art wherein the amino group at the N-terminus appears to the leftand the carboxyl group at the C-terminus appears to the right.

As used herein, the term “amino acid” includes both a naturallyoccurring amino acid and a non-natural amino acid. Unless otherwiseindicated, all amino acids and their residues found in the compoundsdescribed herein can be either in D or L configuration.

The compounds of the invention useful for practicing the methodsdescribed herein may possess one or more chiral centers and so exist ina number of stereoisomeric forms. All stereoisomers and mixtures thereofare included in the scope of the present invention. Racemic compoundsmay either be separated using preparative HPLC and a column with achiral stationary phase or resolved to yield individual enantiomersutilizing methods known to those skilled in the art. In addition, chiralintermediate compounds may be resolved and used to prepare chiralcompounds of the invention.

The compounds described herein may exist in one or more tautomericforms. All tautomers and mixtures thereof are included in the scope ofthe present invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety.

Symbol Meaning Abu α-aminobutyric acid Ac acyl group Aibα-aminoisobutyric acid Ala or A alanine Arg or R arginine Asn or Nasparagine Asp or D aspartic acid Atc

Cha β-cyclohexylalanine sChp

Cys or C cysteine hCys homocysteine Dbu 2,4-diaminobutyric acid Dpr2,3-diaminopropionic acid Gln or Q glutamine Glu or E glutamic acid Glyor G glycine His or H histidine Hyp hydroxyproline Ile or I isoleucineLeu or L leucine Lys or K lysine Met or M methionine 1-Nal(1-naphthyl)-alanine 2-Nal (2-naphthyl)-alanine Nle norleucine Ornornithine Pen penicillamine Phe or F phenylalanine Pro or P proline QAla

Sar sarcosine (N-methylglycine) Ser or S Serine Tle tert-leucine(tert-butyl glycine) TzAla

Thr or T threonine Trp or W tryptopham Tyr or Y tyrosine Val or V valineBHA benzhydrylamine Boc tert-butyloxycarbonyl But tertiary butyl DIPEAN,N-diisopropylethylamine DTT dithiothreitol Fmocfluorenylmethyloxycarbonyl HBTU2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateMCR4 melanocortin-4 receptor Mtt 4-methyltrityl NMP N-methylpyrrolidoneOBut tertiary butoxy OPip 2-phenylisopropyl Pbf2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Trt trityl TIStriisopropylsilane TFA trifluoroacetic acid

Unless otherwise indicated, all abbreviations (e.g. Ala) of amino acidsin this disclosure refer to amino acid residues, i.e. stand for thestructure of —NH—C(R)(R′)—CO—, wherein R and R′ each is, independently,hydrogen or the side chain of an amino acid (e.g., R═CH₃ and R′═H forAla, or R and R′ may be joined to form a ring system).

The designation “Ac” or “NH₂” at a terminus of a polypeptide indicatesthat the corresponding terminus is acylated or amidated, respectively.

The phrase “a covalent bond between amino acid side chains” means thatthe side chains of two amino acid residues in question each includes afunctional group capable of forming a covalent bond with one another.Examples of such bonds include disulfide bridges formed by Cys, hCys, orPen side chains, and amide bonds formed by an amino group of one aminoacid side chain and a carboxy group of another amino acid side chain,such as, e.g. Asp, Glu, Lys, Orn, Dbu, or Dpr. In example embodiments,amino acids can be pairwise selected so as to be able to form covalentbond between their respective side chains. When a covalent bond betweenamino acid side chains is formed, the polypeptide may become cyclized.Such a cyclic polypeptide may be indicated either by a structuralformula or by using the short-hand notation “c( )” or “cyclco( ).” Forexample, “-c(Cys-Cys)-” or “-cyclo(Cys-Cys)-” denotes the structure:

while “-c(Asp-Lys)-” or “-cyclo(Asp-Lys)-” denotes the structure:

“Alkyl” used alone or as part of a larger moiety such as “hydroxyalkyl”,“alkoxyalkyl”, “alkylamine” refers to a straight or branched, saturatedaliphatic group having the specified number of carbons, typically having1 to 12 carbon atoms. More particularly, the aliphatic group may have 1to 8, 1 to 6, or 1 to 4 carbon atoms. This term is exemplified by groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

“Haloalkyl” refers to an alkyl group substituted with one or morehalogen atoms.

“Halogen” and “halo” refer to fluoro, chloro, bromo or iodo.

“Cyano” refers to the group —CN.

“Ph” refers to a phenyl group.

“Carbonyl” refers to a divalent —C(O)— group.

“Aryl” used alone or as part of a larger moiety as in “aralkyl” refersto an aromatic carbocyclic group of from 6 to 18 carbon atoms having asingle ring or multiple condensed rings. The term “aryl” also includesaromatic carbocycle(s) fused to cycloalkyl or heterocycloalkyl groups.Examples of aryl groups include phenyl, benzo[d][1,3]dioxole, naphthyl,phenanthrenyl, and the like.

“Aryloxy” refers to an —OAr group, wherein O is an oxygen atom and Ar isan aryl group as defined above.

“Aralkyl” refers to an alkyl having at least one alkyl hydrogen atomreplaced with an aryl moiety, such as benzyl, —(CH₂)₂phenyl,—(CH₂)₃phenyl, —CH(phenyl)₂, and the like.

“Heteroaryl” used alone or a part of a larger moiety as in“heteroaralkyl” refers to a 5 to 18 membered monocyclic, bicyclic ortricyclic heteroaromatic ring system, containing one to four ringheteroatoms independently selected from nitrogen, oxygen and sulfur. Theterm “heteroaryl” also includes heteroaromatic ring(s) fused tocycloalkyl or heterocycloalkyl groups. Particular examples of heteroarylgroups include optionally substituted pyridyl, pyrrolyl, pyrimidinyl,furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,[2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl,isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl,imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl,quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl,pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl,quinolyl, isoquinolyl, tetrazolyl, 1,2,3,4-tetrahydroquinolyl,1,2,3,4-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl,xanthenyl, benzoquinolyl, and the like.

“Heteroaralkyl” refers to an alkyl having at least one alkyl hydrogenatom replaced with a heteroaryl moiety, such as —CH2-pyridinyl,—CH2-pyrimidinyl, and the like.

“Alkoxy” refers to the group —O—R where R is “alkyl”, “cycloalkyl”,“alkenyl”, or “alkynyl”. Examples of alkoxy groups include for example,methoxy, ethoxy, ethenoxy, and the like.

“Hydroxyalkyl” and “alkoxyalkyl” are alkyl groups substituted withhydroxyl and alkoxy, respectively.

“Amino” means —NH₂; “alkylamine” and “dialkylamine” mean —NHR and —NR₂,respectively, wherein R is an alkyl group. “Cycloalkylamine” and“dicycloalkylamine” mean —NHR and —NR₂, respectively, wherein R is acycloalkyl group. “Cycloalkylalkylamine” means —NHR wherein R is acycloalkylalkyl group. “[Cycloalkylalkyl][alkyl]amine” means —N(R)₂wherein one R is cycloalkylalkyl and the other R is alkyl.

“Acyl” refers to R″—C(O)—, where R″ is H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,aryl, alkylaryl, or substituted alklyaryl, and is indicated in thegeneral formula of a particular embodiment as “Ac”.

Suitable substituents for “alkyl”, “aryl”, or “heteroaryl”, etc., arethose which will form a stable compound of the invention. Examples ofsuitable substituents are those selected from the group consisting ofhalogen, —CN, —OH, —NH₂, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, aryl,heteroaryl, (C₃-C₇)cycloalkyl, (5-7 membered) heterocycloalkyl,—NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, (C₁-C₆)alkoxy,(C₁-C₆)alkoxycarbonyl, —CONH₂, —OCONH₂, —NHCONH₂, —N(C₁-C₆)alkylCONH₂,—N(C₁-C₆)alkylCONH(C₁-C₆)alkyl, —NHCONH(C₁-C₆)alkyl,—NHCON((C₁-C₆)alkyl)₂, —N(C₁-C₆)alkylCON((C₁-C₆)alkyl)₂, —NHC(S)NH₂,—N(C₁-C₆)alkylC(S)NH₂, —N(C₁-C₆)alkylC(S)NH(C₁-C₆)alkyl,—NHC(S)NH(C₁-C₆)alkyl, —NHC(S)N((C₁-C₆)alkyl)₂,—N(C₁-C₆)alkylC(S)N((C₁-C₆)alkyl)₂, —CONH(C₁-C₆)alkyl,—OCONH(C₁-C₆)alkyl —CON((C₁-C₆)alkyl)₂, —C(S)(C₁-C₆)alkyl,—S(O)_(p)(C₁-C₆)alkyl, —S(O)_(p)NH₂, —S(O)_(p)NH(C₁-C₆)alkyl,—S(O)_(p)N((C₁-C₆)alkyl)₂, C₆)alkyl, —OCO(C₁-C₆)alkyl,—C(O)O(C₁-C₆)alkyl, —OC(O)O(C₁-C₆)alkyl, —C(O)H or —CO₂H. Moreparticularly, the substituents are selected from halogen, —CN, —OH,—NH₂, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, phenyl, and(C₃-C₇)cycloalkyl. Within the framework of this invention, said“substitution” is also meant to encompass situations where a hydrogenatom is replaced with a deuterium atom. p is an integer with a value of1 or 2.

Suitable substituents on a substituted Phe include one to fivesubstituents on any aromatic carbons, the substituents being selectedfrom F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂, or —OCH₃. Examplesinclude Phe(2′-F), Phe(2′-CD, Phe(2′-Br), Phe(2′-I), Phe(2′-CN),Phe(2′-CH₃), Phe(2′-OCH₃), Phe(2′-CF₃), Phe(2′-NO₂), Phe(3′-F),Phe(3′-Cl), Phe(3′-Br), Phe(3′-I), Phe(3′-CN), Phe(3′-CH₃),Phe(3′-OCH₃), Phe(3′-CF₃), Phe(3′-NO₂), Phe(4′-F), Phe(4′-Cl),Phe(4′-Br), Phe(4′-I), Phe(4′-CN), Phe(4′-CH₃), Phe(4′-OCH₃),Phe(4′-CF₃), Phe(4′-NO₂), Phe(4′-t-Bu), Phe(2′,4′-diF), Phe(2′,4′-diCl),Phe(2′,4′-diBr), Phe(2′,4′-diI), Phe(2′,4′-di-CN), Phe(2′,4′-di-CH₃),Phe(2′,4′-di-OCH₃), Phe(3′,4′-diF), Phe(3′,4′-diCl), Phe(3′,4′-diBr),Phe(3′,4′-diI), Phe(3′,4′-di-CN), Phe(3′,4′-di-CH₃), Phe(3′,4′-di-OCH₃),Phe(3′,5′-diF), Phe(3′,5′-diCl), Phe(3′,5′-diBr), Phe(3′,5′-diI),Phe(3′,5′-di-CN), Phe(3′,5′-diCH₃), Phe(3′,5′-di-OCH₃), orPhe(3′,4′,5′-triF).

Suitable substituents on a substituted His include one to threesubstituents on any substitutable ring atom, the substituents beingselected from F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂, benzyl, or—OCH₃. Examples include 1-Methyl-Histidine and 3-Methyl-Histidine.

Designation “(amino acid)_(n)” means that an amino acid is repeated ntimes. For example, designation “(Pro)₂” or “(Arg)₃” mean that prolineor arginine residues are repeated, respectively, two or three times.

Pharmaceutically acceptable salts of the polypeptide compounds disclosedherein are included in the present invention. For example, an acid saltof a compound containing an amine or other basic group can be obtainedby reacting the compound with a suitable organic or inorganic acid,resulting in pharmaceutically acceptable anionic salt forms. Examples ofanionic salts include the acetate, benzenesulfonate, benzoate,bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate,chloride, citrate, dihydrochloride, edetate, edisylate, estolate,esylate, fumarate, glyceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate,pamoate, pantothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, tosylate, triethiodide, and trifluoroacetate salts.

Salts of the compounds containing an acidic functional group can beprepared by reacting with a suitable base. Such a pharmaceuticallyacceptable salt can be made with a base which affords a pharmaceuticallyacceptable cation, which includes alkali metal salts (especially sodiumand potassium), alkaline earth metal salts (especially calcium andmagnesium), aluminum salts and ammonium salts, as well as salts madefrom physiologically acceptable organic bases such as trimethylamine,triethylamine, morpholine, pyridine, piperidine, picoline,dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine,bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine,dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine,glucamine, N-methylglucamine, collidine, quinine, quinoline, and basicamino acids such as lysine and arginine.

The disclosed compounds can be administered to the subject inconjunction with an acceptable pharmaceutical carrier as part of apharmaceutical composition. Formulation of the compound to beadministered will vary according to the route of administration selected(e.g., solution, emulsion, capsule). Suitable pharmaceutical carriersmay contain inert ingredients which do not interact with the compound.Standard pharmaceutical formulation techniques can be employed, such asthose described in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa. Suitable pharmaceutical carriers for parenteraladministration include, for example, sterile water, physiologicalsaline, bacteriostatic saline (saline containing about 0.9% mg/ml benzylalcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactateand the like. Methods for encapsulating compositions (such as in acoating of hard gelatin or cyclodextran) are known in the art (Baker, etal., “Controlled Release of Biological Active Agents”, John Wiley andSons, 1986).

As used herein, the phrase “a disorder responsive to the modulation ofthe melanocortin-4 receptor” refers to any disorder that can be treatedby activation (agonizing) or inhibition of MC4R. Examples of suchdisorders will be described in detail below.

As used herein, the term “modulator” refers to compounds which interactwith the target receptor and affects its biological function. Examplesof modulators include full agonists, partial agonists, neutralantagonists, and inverse agonists.

As used herein, the term “agonist” refers to any chemical compound,either naturally occurring or synthetic, that, upon interacting with(e.g., binding to) its target, here, MC4R, raises the signaling activityof MC4R above its basal level. An agonist can be a superagonist (i.e. acompound that is capable of producing a greater maximal response thanthe endogenous agonist for the target receptor, and thus has an efficacyof more than 100%), a full agonist (i.e. a compound that elicits amaximal response following receptor occupation and activation) or apartial agonist (i.e. a compounds that can activate receptors but areunable to elicit the maximal response of the receptor system). Examplesof MC4R agonists will be described in detail below.

As used herein, the term “antagonist” refers to any chemical compound,that, upon interacting with (e.g., binding to) its target, here, MC4R,blocks, in a dose dependent manner, the signaling activity of an agonistcompound with the MC4R.

As used herein, the term “inverse agonist” refers to any chemicalcompound, that, upon interacting with (e.g., binding to) its target,here, MC4R, decreases, in a dose dependent manner, the basal level ofsignaling activity of the MC4R.

As used herein, an “effective amount” refers to an amount of atherapeutic agent or a combination of therapeutic agents that istherapeutically or prophylactically sufficient to treat the targetdisorder. Examples of effective amounts typically range from about0.0001 mg/kg of body weight to about 500 mg/kg of body weight. Anexample range is from about 0.0001 mg/kg of body weight to about 500mg/kg. For example, the effective amount can range from about 0.005mg/kg to about 500 mg/kg. In other examples, the range can be from about0.0001 mg/kg to about 5 mg/kg. In still other examples, effectiveamounts range from about 0.01 mg/kg of body weight to 50 mg/kg of bodyweight, or from 0.01 mg/kg of body weight to 20 mg/kg of body weight.

As used herein, the term “subject” refers to a mammal, preferably ahuman, but can also mean an animal in need of veterinary treatment,e.g., companion animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like).

As used herein, the term “second agent” includes any activepharmaceutical ingredient (API) that, in combination with a peptidedescribed herein, enhances the therapeutic effect produced by a peptidedescribed herein alone or shows synergy with a peptide described herein(i.e. shows the combined effect that is greater than the additiveeffect). As used herein, “an enhanced therapeutic effect” includes animproved therapeutic profile other than synergy. Examples of enhancedtherapeutic effects include lowered effective dose of a peptidedescribed herein, prolonged therapeutic window of a peptide describedherein, etc. One or more second agents can be administered. Examples ofsecond agents will be described in detail below.

A second agent can be administered before, simultaneously with, or afterthe administration of a peptide described herein. Accordingly, a peptidedescribed herein and a second agent can be administered together in asingle formulation or can be administered in separate formulations,e.g., either simultaneously or sequentially. For example, if a peptidedescribed herein and a second agent are administered sequentially inseparate compositions, a peptide described herein can be administeredbefore or after a second therapeutic agent. In addition, a peptidedescribed herein and a second agent may or may not be administered onsimilar dosing schedules. For example, a peptide described herein and asecond therapeutic agent may have different half-lives and/or act ondifferent time-scales such that a peptide described herein isadministered with greater frequency than the second therapeutic agent orvice-versa. Finally, a peptide described herein can be followed by asecond agent, which further enhances therapeutic efficacy, as a resultof the consecutive application of both therapeutic agents. Either apeptide described herein or a second agent can be administered acutelyor chronically.

An effective amount can be achieved in the methods or compositions ofthe invention by co-administering a first amount of a compound having anMC4R modulator activity or a pharmaceutically acceptable salt thereofand a second amount of at least one second agent. In one embodiment, apeptide described herein and second agent are each administered in arespective effective amount (i.e., each in an amount which would betherapeutically effective if administered alone). In another embodiment,a peptide described herein and a second agent are each administered inan amount which alone does not provide a therapeutic effect (asub-therapeutic dose). In yet another embodiment, a peptide describedherein can be administered in an effective amount, while the secondagent is administered in a sub-therapeutic dose. In still anotherembodiment, a peptide described herein can be administered in asub-therapeutic dose, while the second agent is administered in aneffective amount. In example embodiment, a combination of a peptidedescribed herein and a second agent exhibits enhanced therapeutic effector synergy compared to either a peptide described herein or a secondagent alone.

The presence of a synergistic effect can be determined using suitablemethods for assessing drug interaction. Suitable methods include, forexample, the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L.B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loeweadditivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol. Pharmacol.114: 313-326 (1926)), and the median-effect equation (Chou, T. C. andTalalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equationreferred to above can be applied with experimental data to generate acorresponding graph to aid in assessing the effects of the drugcombination. The corresponding graphs associated with the equationsreferred to above are the concentration-effect curve, isobologram curveand combination index curve, respectively.

As used herein “treating” includes achieving, partially orsubstantially, delaying, inhibiting or preventing the progression ofclinical indications related to the target disorder. For example,“treating” includes achieving, partially or substantially, one or moreof the following results: partially or totally reducing the body weight(as measured, for example, by a body mass index, BMI); ameliorating orimproving a clinical symptom or indicators associated with obesity, suchas type-II diabetes, pre-diabetic condition, blood level of haemoglobinA1C (Hb1Ac) above 6%, hyperinsulimenia, hyperlipidemia, insulininsensitivity, glucose intolerance etc; delaying, inhibiting orpreventing the progression of obesity and obesity related indication; orpartially or totally delaying, inhibiting or preventing the onset ordevelopment of obesity or obesity related indication. Delaying,inhibiting or preventing the progression of the obesity includes forexample, delaying, inhibiting or preventing the progression of a subjecthaving normal weight to obesity. The term “treating” further includespartially or totally reducing the risk for coronary artery disease,stroke, and diabetes (e.g. type 2) associated with the metabolicsyndrome as well as ameliorating or improving a clinical symptom orsigns of metabolic syndrome associated with metabolic syndrome, such asany one or more of the five indicators listed above. For example, theterm “treating” includes delaying, inhibiting or preventing theprogression of parameters associated with the metabolic syndrome,including insulin resistance, glucose clearance and parameters ofcardiovascular disease including heart rate and blood pressure, jointdisease, inflammation, sleep apnea, binge eating and other eatingdisorders including bulimia, supportive therapy for weight loss surgeryand supportive weight loss therapy prior to orthopedic surgery.“Prophylactic treatment” refers to treatment before onset of clinicalsymptoms of a target disorder to prevent, inhibit or reduce itsoccurrence.

Disorder Responsive to the Modulation of the MC4R

Examples of disorders responsive to the modulation of MC4R include acuteand chronic inflammatory diseases such as general inflammation,inflammatory bowel disease, brain inflammation, sepsis and septic shock;diseases with an autoimmune component such as rheumatoid arthritis,gouty arthritis, and multiple sclerosis; metabolic diseases and medicalconditions accompanied by weight gain such as obesity, feeding disordersand Prader-Willi Syndrome; metabolic diseases and medical conditionsaccompanied by weight loss such as anorexia, bulimia, AIDS wasting,cachexia, cancer cachexia and wasting in frail elderly; diabetes anddiabetalogical related conditions and complications of diabetes such asretinopathy; neoplastic proliferation such as skin cancer, and prostatecancer; reproductive or sexual medical conditions such as endometriosisand uterine bleeding in women, sexual dysfunction, erectile dysfunctionand decreased sexual response in females; diseases or conditionsresulting from treatment or insult to the organism such as organtransplant rejection, ischemia and reperfusion injury, treatment ofspinal cord injury and to accelerate wound healing, as well as weightloss caused by chemotherapy, radiation therapy, temporary or permanentimmobilization or dialysis; cardiovascular diseases or conditions suchas hemorrhagic shock, cardiogenic shock, hypovolemic shock,cardiovascular disorders and cardiac cachexia; pulmonary diseases orconditions such as acute respiratory distress syndrome, chronicobstructive pulmonary disease, asthma and pulmonary fibrosis; to enhanceimmune tolerance and to combat assaults to the immune system such asthose associated with certain allergies or organ transplant rejection;treatment of dermatological diseases and conditions such as psoriasis,skin pigmentation depletion, acne, keloid formation and skin cancer;behavioral, central nervous system or neuronal conditions and disorderssuch as anxiety, depression, memory and memory dysfunction, modulatingpain perception and treating neuropathic pain; conditions and diseasesassociated with alcohol consumption, alcohol abuse and/or alcoholism;and renal conditions or diseases such as the treatment of renal cachexiaor natriuresis. Additional examples include normalizing or homeostaticactivities in a subject, including thyroxin release, aldosteronesynthesis and release, body temperature, blood pressure, heart rate,vascular tone, brain blood flow, blood glucose levels, bone metabolism,bone formation or development, ovarian weight, placental development,prolactin and FSH secretion, intrauterine fetal growth, parturition,spermatogenesis, sebum and pheromone secretion, neuroprotection andnerve growth as well as modulating motivation, learning and otherbehaviors. Further examples include binge eating, bulimia or othereating disorders.

In example embodiments, the disorders responsive to the modulation ofthe MC4R receptor are type 1 diabetes, type 2 diabetes, obesity, insulinresistance, metabolic syndrome, cardiovascular disease, or low densitylipoprotein/high density lipoprotein/triglyceride imbalance,non-alcoholic fatty liver disease, and disorders of substance abuse.

In example embodiments, the disorders responsive to the modulation ofthe MC4R receptor is type 1 diabetes, type 2 diabetes, obesity, insulinresistance or metabolic syndrome.

Obesity

As used herein, the term “obese” refers to a subject having a body massindex (BMI) of about 30 kg/m² or higher, e.g., a BMI of 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37 kg/m², or more. In particularembodiments, an obese subject has a BMI within the ranges defined as“obese” by the Center for Disease Control. See, URLhttp://www.cdc.gov/obesity/defining.html, last accessed on Oct. 28,2011. For example, in some embodiments, an adult who has a BMI>=30.0kg/m² is obese.

Diabetes and Related Disorders

In example embodiments, subjects to be treated by the methods providedby the invention have or are at increased risk for developing diabetesrelated disorders. “Diabetes-related disorders,” refers to diabetes(including type 1 (OMIM 222100) and type 2 (OMIM 125853)), insulinresistance, and metabolic syndrome.

In example embodiments, the subject to be treated has diabetes (type 1or type 2), insulin resistance, or metabolic syndrome. In exampleembodiments, the disorder is diabetes, e.g. type 2 diabetes. In exampleembodiments, the subject has, or is at increased risk for developing,type 2 diabetes as defined by the World Health Organization and theInternational Diabetes Federation in “Definition and diagnosis ofdiabetes mellitus and intermediate hyperglycaemia,” published in 2006,which is incorporated by reference in its entirety. In exampleembodiments, a diabetic subject exhibits a fasting plasma glucoseof >=126 mg/dL or a 2-hour plasma glucose (2 hours after oraladministration of 75 glucose)>=200 mg/dL. In example embodiments adiabetic or pre-diabetic subject exhibits elevated levels of glycatedhemoglobin, e.g., greater than 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6%,or more of total hemoglobin. In example embodiments, a diabetic orpre-diabetic subject may be identified or further characterized bygenetic polymorphisms (including, for example, polymorphisms leading toaltered expression levels, e.g., elevated or reduced expression levelsand/or variations in coding sequences) in or near one or more of thegenes in Table 1, below:

TABLE 1 Gene/ OMIM Location Locus No. 2q24.1 GPD2 138430 2q31.3 NEUROD1601724 2q36.3 IRS1 147545 3p25.2 PPARG 601487 3q27.2 IGF2BP2 6082894p16.1 WFS1 606201 5q34-q35.2 NIDDM4 608036 6p22.3 CDKAL1 611259 6p21.31HMGA1 600701 6q23.2 ENPP1 173335 7p13 GCK 138079 7q32.1 PAX4 1674138q24.11 SLC30A8 611145 10q25.2-q25.3 TCF7L2 602228 11p15.1 KCNJ11 60093711p15.1 ABCC8 600509 11p11.2 MAPK8IP1 604641 12q24.31 HNF1A 14241013q12.2 IPF1 600733 13q34 IRS2 600797 15q21.3 LIPC 151670 17p13.1 SLC2A4138190 17q12 HNF1B 189907 17q25.3 GCGR 138033 19p13.2 RETN 60556519p13.2 RETN 605565 19q13.2 AKT2 164731 20q12-q13.1 NIDDM3 60369420q13.12 HNF4A 600281 20q13.13 PTPN1 176885

In example embodiments, additional genes that can be used to identify orfurther characterize subjects to be treated by the methods provided bythe invention include FTO (OMIM 610966), JAZF1 (OMIM 606246) and HHEX(OMIM 604420).

In example embodiments, a subject to be treated by the methods providedby the invention has type I diabetes. In example embodiments, a subjectwith type I diabetes is characterized by virtue of a C-peptide assay,e.g., fasting C-peptide levels of less than about 1.0 nmol/L, e.g., lessthan 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nmol/L, or less, e.g.,less than 0.33, 0.25, 0.2, or 0.1 nmol/L. In example embodiments,C-peptide levels are measured after oral glucose challenge (2 hoursafter oral administration of 75 g of glucose) and an increase of lessthan 0.54 nmol/L, e.g., less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25,0.20, 0.15, or 0.10 nmol/L is detected. Impaired fasting glucose(110-125 mg/dL) or impaired glucose tolerance (2-h glucose post-75-gchallenge: 140-199 mg/dL) may be used to identify or furthercharacterize the reduced beta-cell function in subjects with type 1diabetes. In example embodiments, type 1 diabetics are identified orfurther characterized by the presence of autoantibodies to islet cellantigens and/or insulin, e.g., autoantibodies directed to 65 kDa of GAD(OMIM 138275) and/or phosphatase-related IA-2 molecule.

Insulin Resistance

In example embodiments, the disorder is “insulin resistance,” which maybe identified by any means known in the art, and is characterized by areduced ability of insulin to lower blood glucose levels. In exampleembodiments, the insulin resistance is identified or furthercharacterized by the presence of one or more polymorphisms (including,for example, polymorphisms leading to altered expression levels, e.g.,elevated or reduced expression levels as well as coding sequencevariants of gene products, such as proteins) in one or more of thefollowing genes: RETN, PTPN1, TCF1 (OMIM 142410; see, e.g., polymorphism0011), PPP1R3A (OMIM 600917; see, e.g., polymorphisms 0001, 0003), PTPN1(OMIM 176885; see, e.g., polymorphism 0001), ENPP1 (OMIM 173335; see,e.g., polymorphism 0006), IRS1 (OMIM 147545; see, e.g., polymorphism0002), EPHX2 (OMIM 132811; see, e.g., polymorphism 0001), leptin (OMIM164160, see, e.g., polymorphisms 0001 and 0002), leptin receptor (OMIM601007, see, e.g., polymorphisms 0001, 0002, 0004, and 0005), or theinsulin receptor (INSR, OMIM 147670, see, e.g., polymorphisms0001-0037).

Metabolic Syndrome

In example embodiments, the disorder is metabolic syndrome. As usedherein, the term “metabolic syndrome” refers to a group of symptoms thatoccur together and increase the risk for coronary artery disease,stroke, and type 2 diabetes. According to the American Heart Associationand the National Heart, Lung, and Blood Institute, metabolic syndromealso referred to as Syndrome X) is present if a subject has three ormore of the following signs:

1) Blood pressure equal to or higher than 130/85 mmHg;

2) Fasting blood sugar (glucose) equal to or higher than 100 mg/dL;

3) Large waist circumference (length around the waist):

-   -   Men—40 inches or more;    -   Women—35 inches or more;

4) Low HDL cholesterol:

-   -   Men—under 40 mg/dL;    -   Women—under 50 mg/dL;

5) Triglycerides equal to or higher than 150 mg/dL.

Metabolic syndrome can be diagnosed by testing the subject's bloodpressure, blood glucose level, HDL cholesterol level, LDL cholesterollevel, total cholesterol level, and triglyceride level.

In example embodiments the subject has central obesity (waistcircumference >=80 cm for women; >=90 cm for Asian men, including ethnicSouth and Central Americans, and >=94 cm for all other males), BMI>30kg/m², raised triglycerides (>=150 mg/dL, or specific treatment for thislipid abnormality), reduced HDL cholesterol (<40 mg/dL in males, <50mg/dL in females or specific treatment for this lipid abnormality),raised blood pressure (sBP>=130 mmHg or dBP>=85 mmHg or treatment ofpreviously diagnosed hypertension) or raised fasting plasma glucose(FPG>=100 mg/dL or previous type 2 diabetes diagnosis), includingcombinations thereof. In example embodiments, the subject to be treatedby the methods provided by the invention has or is at increased risk formetabolic syndrome, as defined by the International Diabetes Federationin “The IDF consensus worldwide definition of the metabolic syndrome,”published in 2006, which is incorporated by reference in its entirety,i.e., the subject has central obesity (as described above, and/or BMI>30kg/m²) AND any two of raised triglyceries, reduced HDL cholesterol,raised blood pressure, or raised fasting plasma glucose. In exampleembodiments, metabolic syndrome is characterized, or furthercharacterized, by the presence of a mutation at a locus selected from3q27 (see, for example, OMIM 605552) and/or 17p12 (see, for example,OMIM 605572) in the subject.

Disorders Caused by MC4R Mutations

The present invention relates to a method of treating a disorder in asubject suffering from an attenuated response of MC4R to α-melanocortinstimulating hormone (α-MSH). The method comprises administering aneffective amount of an agonist of the melanocortin-4 receptor (MC4R). Inan example embodiment, the subject is a heterozygous carrier of an MC4Rmutation resulting in the attenuated response of MC4R to α-melanocortinstimulating hormone (α-MSH). Because heterozygous carriers retain anability to respond to the natural ligand of MC4R, treatingMC4R-associated disorders in heterozygous carriers by administration ofan MC4R agonist does not rely on the knowledge of the type of the MC4Rmutation.

In one example embodiment, the disorder is obesity, for example,MC4R-associated obesity. In another example embodiment, the disorder ismetabolic syndrome.

Human MC4R gene (hMC4R) is a well-characterized protein encoded by agenomic sequence having GenBank accession number CH471077. Mutations inthe MC4R receptor are an associated cause of severe childhood obesity.The carrier prevalence for MC4R mutations in a juvenile-onset obesepopulation has been noted to be around 2.5% with a highest prevalence of6% among severe obese children. Humans with MC4R mutations show a moreor less similar phenotype as has been described for mice with mutationsin the MC4 receptor gene. Those people show clear hyperphagia,hyperinsulinaemia, increased fat mass, accompanied by lean body mass,bone mineral density and linear growth rate, with no changes in cortisollevels, gonadotropin, thyroid and sex steroid levels. In contrast to MC4receptor deletion, hyperphagia and hyperinsulinaemia tends to subsidewith age in human subjects. Similar to the MC4R knockout mice, thephenotype in heterozygote carriers is intermediate in comparison tohomozygote carriers. The exhibited hyperphagia observed upon a test mealis less severe than that observed in people with a leptin deficiency.The severity of MC4 receptor dysfunction seen in assays in vitro canpredict the amount of food ingested at a test meal by the subjectharboring that particular mutation and correlates with the onset andseverity of the obese phenotype. At least 90 different MC4 receptormutations have been associated with obesity and additional mutations inthe MC4 receptor are likely to be discovered, leading to a similarobesity phenotype.

Examples of the MC4R mutations that cause obesity in humans aredescribed in Farooqi et al., The Journal of Clinical Investigation, July2000, vol. 106 (2), pp. 271-279 and Vaisse et al., The Journal ofClinical Investigation, July 2000, vol. 106(2), pp. 253-262, therelevant portions of which are incorporated herein by reference.

Additional mutations that potentially cause obesity in humans include,R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T,A154D, Y157S, W174C, G181D, F202L, A219V, I226T, G231S, G238D, N240S,C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA, as described inXiang et al., “Pharmacological characterization of 30 humanmelanocortin-4 receptor polymorphisms with the endogenousproopiomelanocortin-derived agonists, synthetic agonists, and theendogenous agouti-related protein antagonist.” Biochemistry, 2010 Jun.8; 49(22):4583-600, the relevant portions of which are incorporatedherein by reference.

Further examples of mutations that potentially cause obesity in humansare those listed in Online Mendelian Inheritance in Man (OMIM), adatabase of human genes and genetic disorders, under the accessionnumber 155541 (MC4R) (more precisely, accession nos.155541.0001-155541.0023) at the URL http://omim.org/entry/155541.Representative examples include 4-BP DEL, NT631; 4-BP INS, NT732;TYR35TER; ASP37VAL; SER58CYS; ILE102SER; ASN274SER; 1-BP INS, 112A; 4-BPDEL, 211CTCT; ILE125LYS; ALA175THR; ILE316SER; TYR287TER; ASN97ASP;15-BP DEL (delta88-92 codons); and SER127LEU. The relevant portions ofthe OMIM database are incorporated herein by reference.

In example embodiments, the MC4R mutation results in retention of theMC4R signaling activity.

Mutations in the genomic sequence encoding MC4R can be detected by themethods that are well known to a person of ordinary skill in the art.For example, the genomic sequence can be cloned using nucleotideprimers, such as e.g., the primers described in Farooqi et al., TheJournal of Clinical Investigation, July 2000, vol. 106 (2), pp. 271-279and Vaisse et al., The Journal of Clinical Investigation, July 2000,vol. 106(2), pp. 253-262, and the cloned sequence analyzed usingcommercially available sequencers and software.

Activity of MC4R can be measured by the methods well known to a personof ordinary skill in the art. For example, cells can be transientlytransfected with the cloned MC4R DNA, the transfected cells contacted byan agonist of MC4R (e.g. α-MSH), and the intracellular level of cAMP,the secondary messenger of MC4R, measured by an electrochemiluminescenceassay described, e.g., in Roubert et al., Journal of Endocrinology(2010) 207, pp. 177-183. A reduction in MC4R signaling can beascertained by comparing the intracellular level of cAMP produced inresponse to a given agonist by a wild type MC4R to that produced by amutant MC4R.

MC4R modulators (e.g. agonists) may also be used to treat patientssuffering from other disorders, such as reduced availability of thenatural agonists of the MC4R. Example of such patients includeindividuals heterozygous or homozygous for mutations in the genesimportant in leptin-dependent pathway (Nature Clinical PracticeEndocrinology and Metabolism, 2006; 2; 6; 318 and NEng J Med: 2007; 356;3; 237), proopiomelanocortin processing (Nature Genetics, 1998, 155;Cell Metabolism, 2006; 3; 135; Annals Acad Med, 2009, 38; 1; 34), ormutations in the genes coding for prohormone convertases.

Modes of Administration

Administration of a compound or pharmaceutically acceptable salt thereofor a composition comprising a compound or pharmaceutical salt of acompound of the invention useful to practice the methods describedherein, can be continuous, hourly, four times daily, three time daily,twice daily, once daily, once every other day, twice weekly, onceweekly, once every two weeks, once a month, or once every two months, orlonger or some other intermittent dosing regimen.

Examples of administration of a compound or composition comprising acompound or pharmaceutical salt of a compound of the invention includeperipheral administration. Examples of peripheral administration includeoral, subcutaneous, intraperitoneal, intramuscular, intravenous, rectal,transdermal, buccal, sublingual, inhalation, pulmonary, or intranasalforms of administration.

Combination Therapy

A peptide described herein can be used for treatment of any of thedisorders responsive to the modulation of MC4R, by administration incombination with one or more other pharmaceutically active compounds(“second agent”). Such combination administration can be by means of asingle dosage form which includes one or more peptides described hereinand one or more second agents, such single dosage forms include atablet, capsule, spray, inhalation powder, injectable liquid, or thelike. Alternatively, combination administration can be by means ofadministration of two different dosage forms, with one dosage formcontaining one or more peptides described herein, and the other dosageform including one or more second agents. In this instance, the dosageforms may be the same or different. Without meaning to limit combinationtherapies, the following exemplifies certain combination therapies whichmay be employed.

A peptide described herein can be combined with one or more secondagents useful in the treatment of various weight and feeding-relateddisorders, such as obesity and/or overweight. In particular, a secondagent can be an anti-obesity drug that affects energy expenditure,glycolysis, gluconeogenesis, glucogenolysis, lipolysis, lipogenesis, fatabsorption, fat storage, fat excretion, hunger and/or satiety and/orcraving mechanisms, appetite/motivation, food intake, orgastrointestinal motility. Drugs that reduce energy intake include, inpart, various pharmacological agents, referred to as anorectic drugs,which are used as adjuncts to behavioral therapy in weight reductionprograms.

Generally, a total dosage of the obesity control agents or medications,when used in combination with one or more peptide described herein canrange from 0.1 to 3,000 mg/day, preferably from about 1 to 1,000 mg/day,and more preferably from about 1 to 200 mg/day in single or 2-4 divideddoses. The exact dose, however, is determined by the attending clinicianand is dependent on such factors as the potency of the compoundadministered, the age, weight, condition, and response of the patient.

One or more peptides described herein can be combined with one or moresecond agents useful in the treatment of diabetes.

One or more peptides described herein can in addition or alternativelyfurther be combined with one or more second agents useful in thetreatment of diseases, disorders and/or conditions associated withobesity and/or overweight, such as insulin resistance; impaired glucosetolerance; type 2 diabetes; metabolic syndrome; dyslipidemia (includinghyperlipidemia); hypertension; heart disorders (e.g. coronary heartdisease, myocardial infarction); cardiovascular disorders; non-alcoholicfatty liver disease (including non-alcoholic steatohepatitis); jointdisorders (including secondary osteoarthritis); gastroesophageal reflux;sleep apnea; atherosclerosis; stroke; macro and micro vascular diseases;steatosis (e.g. in the liver); gall stones; and gallbladder disorders.

Second Agent

The one or more second agents are, for example, selected from:

insulin and insulin analogues;

insulin secretagogues, including sulphonylureas (e.g. glipizide) andprandial glucose regulators (sometimes called “short-actingsecretagogues”), such as meglitinides (e.g. repaglinide andnateglinide);

agents that improve incretin action: an incretin, an incretin mimetic,an agents that improves incretin function e.g. GLP-1, GIP; GLP-1agonists (e.g., exenatide, and liraglutide (VICTOZA)), DPP-4 inhibitors(e.g. vildagliptin, saxagliptin, and sitagliptin)

insulin sensitising agents including peroxisome proliferator activatedreceptor gamma (PPARγ) agonists, such as thiazolidinediones (e.g.pioglitazone and rosiglitazone), and agents with any combination of PPARalpha, gamma and delta activity;

agents that modulate hepatic glucose balance, for example biguanides(e.g. metformin), fructose 1,6-bisphosphatase inhibitors, glycogenphopsphorylase inhibitors, glycogen synthase kinase inhibitors, andglucokinase activators;

agents designed to reduce/slow the absorption of glucose from theintestine, such as alpha-glucosidase inhibitors (e.g. miglitol andacarbose);

agents which antagonize the actions of or reduce secretion of glucagon,such as amylin analogues (e.g. pramlintide);

agents that prevent the reabsorption of glucose by the kidney, such assodium-dependent glucose transporter 2 (SGLT-2) inhibitors (e.g.dapagliflozin);

agents designed to treat the complications of prolonged hyperglycaemia,such as aldose reductase inhibitors (e.g. epalrestat and ranirestat);

agents used to treat complications related to micro-angiopathies;

anti-dyslipidemia agents, such as HMG-CoA reductase inhibitors (statins,e.g. rosuvastatin) and other cholesterol-lowering agents;

PPARα agonists (fibrates, e.g. gemfibrozil and fenofibrate);

bile acid sequestrants (e.g. cholestyramine);

cholesterol absorption inhibitors (e.g. plant sterols (i.e.phytosterols), synthetic inhibitors);

cholesteryl ester transfer protein (CETP) inhibitors; inhibitors of theileal bile acid transport system (IBAT inhibitors);

bile acid binding resins;

nicotinic acid (niacin) and analogues thereof;

anti-oxidants, such as probucol;

omega-3 fatty acids;

antihypertensive agents, including adrenergic receptor antagonists, suchas beta blockers (e.g. atenolol), alpha blockers (e.g. doxazosin), andmixed alpha/beta blockers (e.g. labetalol);

adrenergic receptor agonists, including alpha-2 agonists (e.g.clonidine);

angiotensin converting enzyme (ACE) inhibitors (e.g. lisinopril),calcium channel blockers, such as dihydropyridines (e.g. nifedipine),phenylalkylamines (e.g. verapamil), and benzothiazepines (e.g.diltiazem);

angiotensin II receptor antagonists (e.g. candesartan); aldosteronereceptor antagonists (e.g. eplerenone);

centrally acting adrenergic drugs, such as central alpha agonists (e.g.clonidine); and diuretic agents (e.g. furosemide);

haemostasis modulators, including antithrombotics, such as activators offibrinolysis;

thrombin antagonists;

factor VIIa inhibitors; anticoagulants, such as vitamin K antagonists(e.g. warfarin), heparin and low molecular weight analogues thereof,factor Xa inhibitors, and direct thrombin inhibitors (e.g. argatroban);antiplatelet agents, such as cyclooxygenase inhibitors (e.g. aspirin),adenosine diphosphate (ADP) receptor inhibitors (e.g. clopidogrel),phosphodiesterase inhibitors (e.g. cilostazol), glycoprotein IIB/IIAinhibitors (e.g. tirofiban), and adenosine reuptake inhibitors (e.g.dipyridamole);

anti-obesity agents, such as appetite suppressant (e.g. ephedrine),including noradrenergic agents (e.g. phentermine) and serotonergicagents (e.g. sibutramine), pancreatic lipase inhibitors (e.g. orlistat),microsomal transfer protein (MTP) modulators, diacylglycerolacyltransferase (DGAT) inhibitors, and cannabinoid (CBI)receptor antagonists (e.g. rimonabant);

feeding behavior modifying agents, such as orexin receptor modulatorsand melanin-concentrating hormone (MCH) modulators;

neuropeptide Y (NPY)/NPY receptor modulators;

pyruvate dehydrogenase kinase (PDK) modulators;

serotonin receptor modulators;

leptin/leptin receptor modulators;

ghrelin/ghrelin receptor modulators;

an agent that enhances Beta-cell function;

an agent that stimulates energy expenditure (e.g. beta-adrenergicstimulants, UCP-1 agonists, brown fat modulators and stimulants);

an agent that induces lysis of adipocytes (e.g. an antibody);

nicotine or a nicotine withdrawal aid;

estrogen, a natural or synthetic modulator of an estrogen receptor;

a μ-opioid receptor modulator; and

monoamine transmission-modulating agents, such as selective serotoninreuptake inhibitors (SSRI) (e.g. fluoxetine), noradrenaline reuptakeinhibitors (NARI), noradrenaline-serotonin reuptake inhibitors (SNRI),triple monoamine reuptake blockers (e.g. tesofensine), and monoamineoxidase inhibitors (MAOI) (e.g. toloxatone and amiflamine), or apharmaceutically acceptable salt thereof.

In an example embodiment, an MC4R agonist and a second agent areadministered with the simultaneous, sequential or separateadministration of a very low calorie diets (VLCD) or low-calorie diets(LCD).

Isolated Polypeptides of the Present Invention

In an example embodiment, the isolated polypeptides (e.g., agonists ofMC4R) are those of Formula (I) or a pharmaceutically acceptable saltthereof:

An isolated polypeptide of the following structural Formula (I):

or a pharmaceutically acceptable salt thereof,wherein:

-   -   R¹ is H, or a C1-C6 acyl;    -   R² is, —NR³R⁴, or —OR⁵ wherein R³, R⁴, and R⁵ are each        independently is H or a C1-C6 alkyl;    -   A¹ is an amino acid residue selected from Arg, Lys, Orn, His,        Nle, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu,        or TzAla; or    -   A¹ is a moiety selected from an optionally substituted C1-C12        alkyl, an optionally substituted C6-C18 aryl, an optionally        substituted C5-C18 heteroaryl, an aralkyl wherein the aryl        portion is an optionally substituted C6-C18 aryl, and the alkyl        portion is an optionally substituted C1-C12 alkyl, or a        heteroaralkyl, wherein the heteroaryl portion is an optionally        substituted C5-C18 heteroaryl, and the alkyl portion is an        optionally substituted C1-C12 alkyl;    -   A² and A⁸ is each independently an amino acid residue selected        from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr, wherein A²        and A⁸ are pairwise selected so as to be able to form covalent        bond between their respective side chains;    -   A³ is absent or is an amino acid residue selected from Ala, Tle,        Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar,        Gly, Asn, Aib, or residue Y, wherein Y is an amino acid selected        from amino acids represented by the following structural        formulas

wherein:

-   -   R¹¹ and R¹², each independently, is H, —CH₃, phenyl, or benzyl;    -   R²¹, R²², R²³, and R²⁴, each independently is H, —CH₃, —CF₃,        phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   R³¹, R³², R³³, R³⁴, R⁴¹, R⁴², and R⁴³, each independently is H,        —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   A⁴ is absent or is an amino acid residue selected from Atc, Ala,        QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, an optionally        substituted His, Trp, Tyr, Lys, Arg, sChp, or residue X, where        the X is an amino acid selected from amino acids represented by        the following structural formulas:

wherein:

-   -   R⁵¹ and R⁵², each independently, is H, —CH₃, phenyl, or benzyl;    -   R⁶¹, R⁶², R⁶³, and R⁶⁴, each independently is H, —CH₃, —CF₃,        phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   R⁷¹, R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², and R⁸³, each independently is H,        —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   A⁵ is an optionally substituted Phe, an optionally substituted        1-Nal, or an optionally substituted 2-Nal;    -   A⁶ is Arg; and    -   A⁷ is Trp,

wherein any amino acid residue is either in L- or in D-configuration.

In example embodiments, A³ is absent or is an amino acid residueselected from Ala, Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg,His, Phe, Gln, Sar, Gly, Asn, or Aib; and A⁴ is absent or is an aminoacid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp,Asn, Gln, an optionally substituted His, Trp, Tyr, Lys, Arg, sChp, orresidue X, where the X is an amino acid represented by the followingstructural formula

Values and preferred values of the remainder of the variables are asdefined above and below with respect to Formula (I).

In example embodiments, A³ and A⁴, each independently, is a residue ofan amino acid selected from amino acids represented by the followingstructural formulas:

Values and preferred values of the remainder of the variables are asdefined above and below with respect to Formula (I).

In example embodiments, A³ and A⁴ are not both absent. Values andpreferred values of the remainder of the variables are as defined aboveand below with respect to Formula (I).

In example embodiments, when A⁴ is an amino acid, A³ is not Aib or Gly.Values and preferred values of the remainder of the variables are asdefined above with respect to Formula (I).

In example embodiments, when A⁴ is His and A⁵ is a D-Phe or 2-Nal, A³ isnot a D-amino acid or L-Ala. Values and preferred values of theremainder of the variables are as defined above with respect to Formula(I).

In example embodiments, when A² and A⁸ each is selected from Cys, hCysor Pen, then: (a) when A⁴ is absent, then A³ is not L-His; (b) when A³is absent, then A⁴ is not L-His; and (c) when A⁴ is His, then A³ is notGlu, Leu, or Lys. Values and preferred values of the remainder of thevariables are as defined above with respect to Formula (I).

In example embodiments: 1) A³ and A⁴ are not both absent; 2) when A⁴ isan amino acid, A³ is not Aib or Gly; and 3) when A⁴ is His and A⁵ is aD-Phe or 2-Nal, A³ is not a D-amino acid or L-Ala; 4) when A² and A⁸each is selected from Cys, hCys or Pen, then: (a) when A⁴ is absent,then A³ is not L-His; (b) when A³ is absent, then A⁴ is not L-His; and(c) when A⁴ is His, then A³ is not Glu, Leu, or Lys. Values andpreferred values of the remainder of the variables are as defined abovewith respect to Formula (I).

In another embodiment, the polypeptides of Formula (I), A⁴ is an L-aminoacid. In yet other embodiments, A⁴ is absent. Values and preferredvalues of the remainder of the variables are as defined above withrespect to Formula (I).

In example embodiments, A⁵ can be an optionally substituted 1-Nal or anoptionally substituted 2-Nal, for example, an optionally substitutedD-2-Nal. A⁵ can be substituted at any of the five aromatic carbons witha substituent selected from F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂,or —OCH₃.

In a further embodiment, the polypeptides of Formula (I), A⁵ is anoptionally substituted D-Phe. A⁵ can be substituted at any of the fivearomatic carbons with a substituent selected from F, Cl, Br, I, —CH₃,—OH, —CN, amine, —NO₂, or —OCH₃. Suitable examples of A⁵ include, butare not limited to, a D-amino acid residue selected from: Phe,Phe(2′-F), Phe(2′-Cl), Phe(2′-Br), Phe(2′-I), Phe(2′-CN), Phe(2′-CH₃),Phe(2′-OCH₃), Phe(2′-CF₃), Phe(2′-NO₂), Phe(3′-F), Phe(3′-Cl),Phe(3′-Br), Phe(3′-I), Phe(3′-CN), Phe(3′-CH₃), Phe(3′-OCH₃),Phe(3′-CF₃), Phe(3′-NO₂), Phe(4′-F), Phe(4′-Cl), Phe(4′-Br), Phe(4′-I),Phe(4′-CN), Phe(4′-CH₃), Phe(4′-OCH₃), Phe(4′-CF₃), Phe(4′-NO₂),Phe(4′-t-Bu), Phe(2′,4′-diF), Phe(2′,4′-diCl), Phe(2′,4′-diBr),Phe(2′,4′-diI), Phe(2′,4′-di-CN), Phe(2′,4′-di-CH₃), Phe(2′,4′-di-OCH₃),Phe(3′,4′-diF), Phe(3′,4′-diCl), Phe(3′,4′-diBr), Phe(3′,4′-diI),Phe(3′,4′-di-CN), Phe(3′,4′-di-CH₃), Phe(3′,4′-di-OCH₃), Phe(3′,5′-diF),Phe(3′,5′-diCl), Phe(3′,5′-diBr), Phe(3′,5′-diI), Phe(3′,5′-di-CN),Phe(3′,5′-diCH₃), Phe(3′,5′-di-OCH₃), or Phe(3′,4′,5′-triF). Values andpreferred values of the remainder of the variables are as defined abovewith respect to Formula (I).

In a further embodiment, the polypeptides of Formula (I), A⁵ is anoptionally substituted D-2-Nal. A⁵ can be substituted at any of the fivearomatic carbons with a substituent selected from F, Cl, Br, I, —CH₃,—OH, —CN, amine, —NO₂, or —OCH₃.

In yet another embodiment, the polypeptides of Formula (I), A⁴ is His,optionally substituted at any substitutable position with a substituentselected from F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂, benzyl or—OCH₃. Values and preferred values of the remainder of the variables areas defined above with respect to Formula (I).

In a particular embodiment, the compounds of the present invention arethose polypeptides of Formula (I) that possess EC₅₀ with respect to MC4Rfrom about 0.01 nM to about 10 nM, for example 0.01-3 nM, whilepossessing the ratio of EC₅₀(MC1R)/EC₅₀(MC4R) of at least 10.

In another embodiment, the polypeptides of the present invention includea polypeptide represented by any one of the following structuralformulas:

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the polypeptides of the present inventioninclude any one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude the polypeptide represented by any one of the followingstructural formulas:

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the polypeptides of the present inventioninclude a polypeptide represented by formula (I), wherein A⁴ is an aminoacid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp,Asn, Gln, a substituted His, Trp, Tyr, Lys, Arg, sChp, or residue X.Examples of such peptides include peptides represented by any one of thefollowing structural formulas:

(SEQ ID NO: 36) Ac-Arg-cyclo[Cys-D-Ala-His(3-Me)-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 37)Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp- Cys]-NH₂; (SEQ ID NO: 9)Ac-Arg-cyclo[Cys-D-Ala-Trp-D-Phe-Arg-Trp-Cys]- NH₂;  (SEQ ID NO: 8)Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 7)Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 38)Ac-Arg-cyclo[Cys-D-Ala-Arg-D-Phe-Arg-Trp-Cys]-NH₂;   (SEQ ID NO: 39)Ac-Arg-cyclo[Cys-D-Ala-Tyr-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 40)Ac-Arg-cyclo[Cys-D-Ala-D-Pro-D-Phe-Arg-Trp-Cys]- NH₂;  (SEQ ID NO: 2)Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 4)Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe(p-F)-Arg-Trp- Cys]-NH₂; (SEQ ID NO: 41) Ac-Arg-cyclo[Cys-D-Ala-Atc-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 42) Ac-Arg-cyclo[Cys-D-Ala-QAla-D-Phe-Arg-Trp-Cys]- NH₂; (SEQ ID NO: 43) Ac-Arg-cyclo[Cys-D-Ala-sChp-D-Phe-Arg-Trp-Cys]- NH₂;  or(SEQ ID NO: 44) Ac-Arg-cyclo[Cys-D-Ala-X-D-Phe-Arg-Trp-Cys]-NH₂, or a pharmaceutically acceptable salt thereof.

In example embodiments, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 15) Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 14) Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 45) Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Pen]-NH₂;(SEQ ID NO: 26) Ac-Arg-cyclo[Glu-D-Ala-D-Phe-Arg-Trp-Dpr]-NH₂; (SEQ ID NO: 27) Ac-Arg-cyclo[Glu-Ala-D-Phe-Arg-Trp-Dpr]-NH₂; (SEQ ID NO: 46) Ac-Arg-cyclo[hCys-Aib-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 47) Ac-Arg-cyclo[hCys-Sar-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 48) Ac-Arg-cyclo[hCys-Val-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 49) Ac-Arg-cyclo[hCys-D-Val-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 50) Ac-Arg-cyclo[hCys-Gln-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 51) Ac-Arg-cyclo[hCys-D-Gln-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 52) Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Pen]-NH₂;(SEQ ID NO: 53) Ac-Arg-cyclo[D-Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂;(SEQ ID NO: 17) Ac-Arg-cyclo[Cys-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂; (SEQ ID NO: 54) Ac-Arg-cyclo[Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂; (SEQ ID NO: 55) Ac-Arg-cyclo[D-hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 20) Ac-Arg-cyclo[hCys-Pro-D-Phe-Arg-Trp-Cys]-NH₂; or(SEQ ID NO: 56) Ac-Arg-cyclo[hCys-D-Pro-D-Phe-Arg-Trp-Cys]-NH₂,or a pharmaceutically acceptable salt thereof.

In another embodiment, the polypeptides of the present invention includepolypeptides represented by formula (I), wherein A³ is an amino acidresidue selected from Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg,His, Phe, Gln, Sar, Gly, Asn, or Aib; and A⁴ is an amino acid residueselected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, asubstituted His, Trp, Tyr, Lys, Arg, sChp, or residue X. Examples ofsuch polypeptides are polypeptides represented by any one of thefollowing structural formulas:

Ac-Arg-cyclo[Cys-Val-Gln-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 57)

Ac-Arg-cyclo[Cys-D-Val-Gln-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 11) or

Ac-Arg-cyclo[Cys-D-Val-His(1-Me)-D-Phe-Arg-Trp-Cys]-NH₂, (SEQ ID NO: 58)

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 59) Ac-TzAla-cyclo[Cys-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂;  or(SEQ ID NO: 60) Ac-Glu-cyclo[Cys-Ala-His-D-Phe-Arg-Trp-Cys]-NH₂, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 37)

(SEQ ID NO: 8) Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂;  or(SEQ ID NO: 7) Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-NH₂, or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 61) Ac-Arg-cyclo[Cys-D-Leu-His-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 62) Ac-Arg-cyclo[Cys-D-Ile-His-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 63) Ac-Arg-cyclo[Cys-D-Tle-His-D-Phe-Arg-Trp-Cys]-NH₂;  or(SEQ ID NO: 10) Ac-Arg-cyclo[Cys-D-Val-His-D-Phe-Arg-Trp-Cys]-NH₂, or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 64) Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-2-Nal-Arg-Trp- Cys]-NH₂; (SEQ ID NO: 65)Ac-Arg-cyclo[Cys-D-Ala-Gln-D-2-Nal-Arg-Trp-Cys]- NH₂;  or(SEQ ID NO: 66) Ac-Arg-cyclo[Cys-D-Ala-Asn-D-2-Nal-Arg-Trp-Cys]- NH₂, or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 67) Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp- Cys]-OH; (SEQ ID NO: 68)Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-OH;  or (SEQ ID NO: 69)Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-OH, or a pharmaceutically acceptable salt thereof.

EXEMPLIFICATION Peptide Synthesis

The peptides of this invention were prepared by conventional solid phasepeptide synthesis. The peptide chain was elongated in a step-wise mannerstarting with its C-terminal end amino acid derivative coupled onto anappropriately selected solid support resin known to be suitable forpeptide synthesis. For the synthesis of peptide with a C-terminal amidefunction, Rink amide MBHA resin was employed as solid support. For thesynthesis of peptides with the C-terminal free carboxyl function, resinssuch as 2-chlorotrityl chloride resin, Wang, or Merrifield resin may beutilized that form an ester bond with the Fmoc-amino acid. Most of theseester linked Fmoc-amino acid-resin types are commercially available fromvarious sources and generally used when feasible.

Synthesis of Disulfide-Cyclized Peptides

The linear derivative of a disulfide cyclic peptides amide was assembledusing Fmoc chemistry on a solid-phase peptide synthesizer. The Fmoc-Rinkamide resin was placed in a reaction vessel and swollen with NMP. It wasthen treated with 20% piperidine in NMP for 15 minutes, followed by 3washes of NMP. The resin was tested for positive Kaiser's test (Kaiser,E., Colescot, R. L., Bossinge, C. D. & Cook, P. I. Anal. Biochem., 1990,34: 595-598). It was resuspended in NMP and mixed with the requiredfirst C-terminal Fmoc-amino acid derivative and HOBt. The couplingreaction was started by the addition of HBTU reagent and DIPEA. Aftermixing for 2-3 hours, the completion of coupling was confirmed by anegative Kaiser's test on a small aliquot of the resin withdrawn fromthe reaction mixture. The resin was then washed three times with NMP.Thereafter, the Fmoc group was removed as described earlier and thewhole cycle repeated with the second C-terminal Fmoc-amino acidderivative as described. The same cycle of reactions was repeatedsequentially with each of the incoming amino acid. The chloranil colortest (Vojkovsky, T. Pept. Res., 1995, 8: 236-237) was used instead ofKaiser's test for positive testing of Fmoc deprotection from the prolineresidue in the peptide sequence as well for testing completion ofcoupling of an amino acid to proline (a negative chloranil test). Incase of peptides with N-terminal acetyl group, the Fmoc deprotectedpeptide resin was treated for 10 minutes with acetic anhydride andpyridine. The resin after testing negative for Kaiser's test was washedwith NMP, dichloromethane and dried in vacuo. The Fmoc-amino acidderivatives were used for the synthesis of these peptides. Thetrifunctional amino acid derivatives used were the following:Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-hCys(Trt)-OH, Fmoc-Pen(Trt)-OH,Fmoc-Tyr(But)-OH, Fmoc-His(1-Me)-OH, Fmoc-His(3-Me)-OH, andFmoc-Glu(OBut)-OH.

To cleave the peptide off the resin as well as to deprotect the sidechain functions, the peptide resin was taken in: 2% TIS/5% water/5%(w/v) DTT/88% TFA. The solution was allowed to mix for 3.5 hours andthen filtered. The filtrate was mixed with cold anhydrous ethyl ether.The precipitate was collected by centrifugation. The solvent wasdecanted and the peptide pellet was re-suspended in fresh ether. Theether workup was repeated two more times. The peptide was dried invacuo. The crude linear peptide product was diluted to a concentrationof 2 mg/mL in 5% acetic acid and 0.5 M iodine/methanol was addeddropwise with vigorous stirring until a persistent pale yellow color ofthe solution was achieved. The solution was stirred for additional 10minutes. Excess iodine was then quenched by adding 1 M sodiumthiosulfate under mixing until the mixture was rendered colorless. Thecyclized peptide solution was lyophilized and the crude powder purifiedby preparative HPLC using a reversed-phase C-18 column. The purifiedproduct fractions were pooled and lyophilized. The peptides wereanalyzed by mass spectrometry using electrospray ionization techniqueand identified to correct mass.

Synthesis of Lactm-Cyclized Peptides

The cyclic lactam peptides were also synthesized by standard solid phasepeptide synthesis methods. For peptides with a C-terminus Dpr, anFmoc-Dpr(Mtt)-BHA resin was transferred to a solid phase peptidesynthesizer reactor. The Fmoc group, was removed as described above andthe next Fmoc-protected amino acid, such as for exampleFmoc-Trp(Boc)-OH, was coupled to the resin through standard couplingprocedures. The Fmoc protective group was removed and the remainingamino acids added individually in the correct sequence, by repeatingcoupling and deprotection procedures until the amino acid sequence wascompleted. For glutamic acid, coupling Fmoc-Glu(OPip) was employed. Thefully assembled peptide was then acetylated at the N-terminus as permethod described earlier for the disulfide series of peptides. Theorthogonally protected side chains were then removed. For example, apeptide resin with either an orthogonally protected side chain of Glu as2-phenylisopropyl (OPip) ester or Dpr as 4-methyltrityl (Mtt), werecleaved by treatment with 1% TFA in dicholoromethane. The deprotectedpeptide resin was suspended in NMP, and treated with HBTU/DIPEA. Aftercyclization (a negative Kaiser's test), the peptide-resin was washedwith DCM and dried. The cyclic peptide was cleaved from the resin alongwith any remaining protective groups using trifluoroacetic acid (TFA) inthe presence of water and 1,2-ethanedithiol (EDT). The product wascollected by precipitation upon the addition of cold anhydrous ether andcollected by centrifugation. Final purification was by reversed phaseHPLC using a reversed phase C-18 column. The purified peptide collectedby lyophilization and analyzed for its mass by mass spectrometry usingelectron spray methodology.

Examples of the compounds of the present invention are provided in Table2.

TABLE 2 Example Compounds of the Invention Com- pound  1Ac-Arg-cyclo[Cys-D-Leu-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 61)  2Ac-Arg-cyclo[Cys-D-Ile-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 62)  3Ac-Arg-cyclo[Cys-D-Tle-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 63) Tle =t-butyl glycine  4 Ac-Arg-cyclo[Cys-D-Val-His-D-Phe-Arg-Trp-Cys]-NH₂(SEQ ID NO: 10)  5Ac-Arg-cyclo[Cys-D-Ala-His(3-Me)-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 36) 6 Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO:37)  7 Ac-Arg-cyclo[Cys-D-Ala-Trp-D-Phe-Arg-Trp-Cys ]-NH₂ (SEQ ID NO: 9) 8 Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 8)  9Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 7) 10Ac-Arg-cyclo[Cys-D-Ala-Arg-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 38) 11Ac-Arg-cyclo[Cys-D-Ala-Tyr-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 39) 12Ac-Arg-cyclo [Cys-D-Ala-D-Pro-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 40) 13Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 2) 14Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe(p-F)-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 4) S1Ac-Arg-cyclo [Cys-D-Ala-Atc-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 41)

S2 Ac-Arg-cyclo [Cys-D-Ala-QAla-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 42)

S3 Ac-Arg-cyclo [Cys-D-Ala-sChp-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 43)

S4 Ac-Arg-cyclo [Cys-D-Ala-X-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 44)

15 Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 15) 16Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 14) 17Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Pen]-NH₂ (SEQ ID NO: 45) 18Ac-Arg-cyclo[Glu-D-Ala-D-Phe-Arg-Trp-Dpr]-NH₂ (SEQ ID NO: 26) 19Ac-Arg-cyclo[Glu-Ala-D-Phe-Arg-Trp-Dpr]-NH₂ (SEQ ID NO: 27) S5Ac-Arg-cyclo[hCys-Aib-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 46) S6Ac-Arg-cyclo [hCys-Sar-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 47) S7Ac-Arg-cyclo [hCys-Val-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 48) S8Ac-Arg-cyclo [hCys-D-Val-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 49) S9Ac-Arg-cyclo [hCys-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 50) S10Ac-Arg-cyclo [hCys-D-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 51) S11Ac-Arg-cyclo [hCys-Ala-D-Phe-Arg-Trp-Pen]-NH₂ (SEQ ID NO: 52) S12Ac-Arg-cyclo [D-Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂ (SEQ ID NO: 53) S13Ac-Arg-cyclo [Cys-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂ (SEQ ID NO: 17) S14Ac-Arg-cyclo [Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂ (SEQ ID NO: 54) S15Ac-Arg-cyclo [D-hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 55) D1Ac-Arg-cyclo [hCys-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 20) D2Ac-Arg-cyclo [hCys-D-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 56) 20Ac-Arg-cyclo[Cys-Val-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 57) 21Ac-Arg-cyclo[Cys-D-Val-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 11) D3Ac-Arg-cyclo[Cys-D-Val-His(1-Me)-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 58)D4 Ac-TzAla-cyclo[Cys-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 59)

22 Ac-Glu-cyclo[Cys-Ala-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 60)Radioligand Binding Assays:

Receptor binding assays for determining the binding constant (K_(d)) orinhibition concentration (IC₅₀) for displacing a radio-labeled ligandfrom the receptor of a cyclic peptide of the invention may be conductedby any means known in the art.

As an example, the cell membrane preparations for a binding assay areprepared from CHO-K1 cells transfected to stably express hMC receptorsubtypes 1, 3, 4 or 5. Competitive inhibition of[125I](Tyr2)-(Nle4-D-Phe7)-alpha-MSH ([125I]-NDP-α-MSH binding iscarried out in polypropylene 96 well plates. Briefly, the cell membranes(1-10 μg protein/well), prepared as described above, is incubated in 50mM Tris-HCl at pH 7.4 containing 0.2% BSA, 5 mM MgCl2, 1 mM CaCl2 and0.1 mg/mL bacitracin, with increasing concentrations of the testcompound and 0.1-0.3 nM [125I]-NDP-α-MSH for approximately 120 minutesat 37° C. Bound [125I]-NDP-α-MSH ligand is separated from free[125I]-NDP-α-MSH by filtration through GF/C glass fiber filter plates(Unifilter®, Meriden, Conn., USA) presoaked with 0.1% (w/v)polyethylenimine (PEI). Filters are washed three times with 50 mMTris-HCl at pH 7.4 at a temperature of approximately 0-4° C. and thenassayed for radioactivity. The binding data are analyzed bycomputer-assisted non-linear regression analysis.

Cyclic AMP Stimulation Assay

Functional assays to determine agonist or antagonist status of a cyclicpeptide of the invention may be conducted by any means known in the art.

Electrochemiluminescence (ECL) Assay

Stimulation of intracellular cyclic AMP (cAMP) levels by the peptides isdetermined in a dose dependent manner by an electrochemiluminescence(ECL) assay (Meso Scale Discovery, Gaithersburg, Md., USA; referred tohereinafter as “MSD”). Briefly, the CHO-K1 cells stably expressing thehMC receptor subtypes are suspended in RMPI 1640® assay buffer (RMPI1640 buffer contains 0.5 mM IBMX, and 0.2% protein cocktail (MSD blockerA)). About 7,000 cells/well of the transgenic CHO-K1 cells stablyexpressing hMC receptor subtypes 1, 3, 4 or 5 are dispensed in 384-wellMulti-Array plates (MSD) containing integrated carbon electrodes andcoated with anti-cAMP antibody. Increasing concentrations of the testcompounds are added and the cells are incubated for approximately 40minutes at 37° C. A cell lysis buffer (HEPES-buffered saline solutionwith MgCl2 and Triton X-100® at pH 7.3) containing 0.2% protein cocktailand 2.5 nM TAG™ ruthenium-labeled cAMP (MSD) is added and the cells areincubated for approximately 90 minutes at room temperature. At the endof the second incubation period, the read buffer (Tris-buffered solutioncontaining an ECL co-reactant and Triton X-100 at pH 7.8) is added andthe cAMP levels in the cell lysates are immediately determined by ECLdetection with a Sector Imager 6000 Reader® (MSD). Data are analyzedusing a computer-assisted non-linear regression analysis (XL fit; IDBS)and reported as either an EC50 value. The EC50 represents theconcentration of an agonist compound needed to obtain 50% of the maximumreaction response, e.g., 50% of the maximum level of cAMP as determinedusing the assay described above.

cAMP Measurement Assay

Human MC4-R transfected cells are grown to confluence in 96 well plates(plating approximately 250,000 cells per well). The cells are treated intriplicate sets with 0.2 mM isobutylmethylxanthine (IBMX) and gradedconcentrations of the peptide or alternatively the peptide in thepresence of 20 nM NDP-MSH. Cells similarly treated but with only 20 nMNDP-MSH serve as positive controls in a volume of 200 μL. A buffer blankserving as a negative control is also included. After incubation of onehour at 37° C., the cells are lysed by the addition of 50 μL of a celllysis buffer. Total cAMP accumulated in 250 μL of this incubation mediumis quantitated using a commercially available low pH cAMP assay kit(Amersham BioSciences) as per procedure specified by the kit supplier. Apeptide showing cAMP accumulation in the range same or higher than thealpha-MSH as positive control is considered to be an agonist. The datafor agonist is plotted and curve fitted to determine the EC50 value. Apeptide showing accumulation in the same range as the negative control(buffer blank in the absence of alpha-MSH) is ineffective at the testconcentration. A peptide showing attenuated accumulation is consideredto be an antagonist if there is inhibition in cAMP when alpha-MSH isalso present in the assay. Similar assay can be performed with hMC-1R,hMC-3R, and hMC-5R cells.

cAMP Accumulation Measurement Via a β-Galactosidase (β-Gal) ReporterSystem

A chemiluminescence readout system that uses an enzyme fragmentcomplementation (EFC) system with β-galactosidase (β-Gal) as thefunctional reporter system was used. This assay system for variousmelanocortin receptor systems is commercially available (cAMP HunterGPCR assay system, Discoverx Corp, Fremont, Calif.). This assay utilizesthe β-Gal enzyme that is split into two complementary portions; EA forEnzyme Acceptor and ED for Enzyme Donor. In the assay, the ED portionfused to cAMP is made to compete with cAMP generated by cells forbinding to a cAMP-specific antibody. The EA is then added to form activeβ-Gal with any unbound ED-cAMP. This active enzyme then converts achemiluminescent substrate to generate an output signal that is recordedon a standard microplate reader.

Briefly, 10000 cells per well are plated overnight and each well (cellsincubated with 10 μl assay buffer) is then incubated with 4× serialconcentration of the test compound in the cell assay buffer (5 μL) andcAMP antibody reagent (5 μL) for 30 min at 37° C. The cell lysis buffer(20 μL) containing ED-cAMP coupled enzyme fragment and the reportersubstrate (Emerald II-Galacton Star, 5:1) is then added and incubatedfor 60 min at room temperature. Next, 20 μL of EA β-Gal fragment reagentis added. After further incubation for 120 min at room temperature, thechemiluminescence is measured by a plate reader (Envision), and the dataused to calculate EC50 values for the test peptide.

The results are presented in Table 3.

TABLE 3 EC50 (nM) values of example compounds of the invention cAMPAssay (EC-50) Ratios of EC50 values Com- MC MC MC pound MC1R MC3R MC4RMC5R ¼ ¾ 5/4 1 0.47 0.79 0.70 91 0.68 1.13 130 2 0.69 0.96 1 420 0.690.96 420 3 1 0.7 0.7 672 1 1 930 4 1.25 1.59 1.34 782 0.93 1.19 584 54.1 405.8 1.15 1085 4 350 945 6 30.4 4.3 0.7 467 40 6 662 7 273 >10 uM34 259 8 >290 7 8 71 8.6 1.6 255 40 5 155 9 248 81 3 1490 90 30 530 106.2 3.9 2.7 2.31 1.45 11 300.9 >1000 45.1 6.67 >22.2 12 13 280 >10 uM 56707 5 >200 13 14 169 >10 uM 24 283 7 >400 12 15 4 1 0.26 42 15 3.8 16116 888 3158 7.5 >10000 120 420 >1338 17 195 233 13.7 2181 15 17 159 221.7 9.9 <0.5 1282 >3 >20 >2563

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An isolated polypeptide of the followingstructural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —H, or aC1-C6 acyl; R² is —NR³R⁴ or —OR⁵, wherein R³, R⁴, and R⁵ are eachindependently H or a C1-C6 alkyl; A¹ is an amino acid residue selectedfrom Arg, Lys, Orn, His, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln,Asn, Asp, Glu, or TzAla; or A¹ is a moiety selected from an optionallysubstituted C1-C12 alkyl, an optionally substituted C6-C18 aryl, anoptionally substituted C5-C18 heteroaryl, an aralkyl wherein the arylportion is an optionally substituted C6-C18 aryl, and the alkyl portionis an optionally substituted C1-C12 alkyl, or a heteroaralkyl, whereinthe heteroaryl portion is an optionally substituted C5-C18 heteroaryl,and the alkyl portion is an optionally substituted C1-C12 alkyl; or A¹is absent; or A² and A⁸ is each independently an amino acid residueselected from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr, whereinA² and A⁸ are pairwise selected so as to be able to form covalent bondbetween their respective side chains; A³ is an amino acid residueselected from Ala, Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg,His, Phe, Gln, Sar, Gly, Asn, Aib, or A³ is absent, or A³ is residue Y,wherein Y is an amino acid selected from amino acids represented by thefollowing structural formulas

wherein: R¹¹ and R¹², each independently, is H, —CH₃, phenyl, or benzyl;R²¹, R²², R²³, and R²⁴, each independently is H, —CH₃, —CF₃, phenyl,benzyl, F, Cl, Br, I, —OCH₃, or —OH; R³¹, R³², R³³, R³⁴, R⁴¹, R⁴², andR⁴³, each independently is H, —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I,—OCH₃, or —OH; A⁴ is absent or is an amino acid residue selected fromAtc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, an optionallysubstituted His, Trp, Tyr, Lys, Arg, sChp, or residue X, where the X isan amino acid selected from amino acids represented by the followingstructural formulas:

wherein: R⁵¹ and R⁵², each independently, is H, —CH₃, phenyl, or benzyl;R⁶¹, R⁶², R⁶³, and R⁶⁴, each independently is H, —CH₃, —CF₃, phenyl,benzyl, F, Cl, Br, I, —OCH₃, or —OH; R⁷¹, R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², andR⁸³, each independently is H, —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I,—OCH₃, or —OH; A⁵ is an optionally substituted Phe, an optionallysubstituted 1-Nal, or an optionally substituted 2-Nal; A⁶ is Arg; and A⁷is Trp, wherein any amino acid residue is either in L- or inD-configuration, provided that: 1) A³ and A⁴ are not both absent; 2)when A⁴ is an amino acid, A³ is not Aib or Gly; 3) when A⁴ isunsubstituted His and A⁵ is unsubstituted D-Phe or 2-Nal, A³ is not aD-amino acid or L-Ala; 4) when A² and A⁸ each is selected from Cys, hCysor Pen, then: (a) when A⁴ is absent, then A³ is not L-His; (b) when A³is absent, then A⁴ is not L-His; (c) when A⁴ is His, then A³ is not Glu,Leu, or Lys; and 5) when A² is Asp, Glu, Lys, or Orn, then A⁸ is notAsp, Glu, Lys, or Orn.
 2. An isolated polypeptide of claim 1, wherein:R¹ is —H, or a C1-C6 acyl; R² is —NR³R⁴, or —OR⁵ wherein R³, R⁴, and R⁵are each independently is H or a C1-C6 alkyl; A¹ is absent; or A¹ is anamino acid residue selected from Arg, Lys, Orn, His, Phe, Val, Leu, Trp,Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu, or TzAla; or A¹ is a moietyselected from an optionally substituted C1-C12 alkyl, an optionallysubstituted C6-C18 aryl, an optionally substituted C5-C18 heteroaryl, anaralkyl wherein the aryl portion is an optionally substituted C6-C18aryl, and the alkyl portion is an optionally substituted C1-C12 alkyl,or a heteroaralkyl, wherein the heteroaryl portion is an optionallysubstituted C5-C18 heteroaryl, and the alkyl portion is an optionallysubstituted C1-C12 alkyl; A² and A⁸ is each independently an amino acidresidue selected from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr,wherein A² and A⁸ are pairwise selected so as to be able to formcovalent bond between their respective side chains; A³ is absent or isan amino acid residue selected from Ala, Tle, Val, Leu, Be, Cha, Pro,Ser, Thr, Lys, Arg, His, Phe, Gln, Sar, Gly, Asn, or Aib; A⁴ is absentor is an amino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser,Thr, Pro, Hyp, Asn, Gln, an optionally substituted His, Trp, Tyr, Lys,Arg, sChp, or residue X, where the X is an amino acid represented by thefollowing structural formula

A⁵ is an optionally substituted Phe, an optionally substituted 1-Nal, oran optionally substituted 2-Nal; A⁶ is Arg; and A⁷ is Trp, wherein anyamino acid residue is either in L- or in D-configuration.
 3. Thepolypeptide of claim 1, wherein A³ is a D-amino acid.
 4. The polypeptideof claim 1, wherein A⁴ is an L-amino acid.
 5. The polypeptide of claim1, wherein A⁵ is an optionally substituted D-Phe.
 6. The polypeptide ofclaim 1, wherein A⁵ is a D-amino acid residue selected from: Phe,Phe(2′-F), Phe(2′-Cl), Phe(2′-Br), Phe(2′-I), Phe(2′-CN), Phe(2′-CH₃),Phe(2′-OCH₃), Phe(2′-CF₃), Phe(2′-NO₂), Phe(3′-F), Phe(3′-Cl),Phe(3′-Br), Phe(3′-I), Phe(3′-CN), Phe(3′-CH₃), Phe(3′-OCH₃),Phe(3′-CF₃), Phe(3′-NO₂), Phe(4′-F), Phe(4′-Cl), Phe(4′-Br), Phe(4′-I),Phe(4′-CN), Phe(4′-CH₃), Phe(4′-OCH₃), Phe(4′-CF₃), Phe(4′-NO₂),Phe(4′-t-Bu), Phe(2′,4′-diF), Phe(2′,4′-diCl), Phe(2′,4′-diBr),Phe(2′,4′-diI), Phe(2′,4′-di-CN), Phe(2′,4′-di-CH₃), Phe(2′,4′-di-OCH₃),Phe(3′,4′-diF), Phe(3′,4′-diCl), Phe(3′,4′-diBr), Phe(3′,4′-diI),Phe(3′,4′-di-CN), Phe(3′,4′-di-CH₃), Phe(3′,4′-di-OCH₃), Phe(3′,5′-diF),Phe(3′,5′-diCl), Phe(3′,5′-diBr), Phe(3′,5′-diI), Phe(3′, 5′-di-CN),Phe(3′,5′-diCH₃), Phe(3′,5′-di-OCH₃), or Phe(3′,4′,5′-triF).
 7. Thepolypeptide of claim 1, represented by any one of the followingstructural formulas:

or a pharmaceutically acceptable salt thereof.
 8. The polypeptide ofclaim 1, wherein A⁴ is an amino acid residue selected from Atc, Ala,QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, a substituted His, Trp,Tyr, Lys, Arg, sChp, and residue X.
 9. The polypeptide of claim 8,represented by any one of the following structural formulas:(SEQ ID NO: 36) Ac-Arg-cyclo[Cys-D-Ala-His(3-Me)-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 37)Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp- Cys]-NH₂; (SEQ ID NO: 9) Ac-Arg-cyclo[Cys-D-Ala-Trp-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 8) Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 7) Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 38) Ac-Arg-cyclo[Cys-D-Ala-Arg-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 39) Ac-Arg-cyclo[Cys-D-Ala-Tyr-D-Phe-Arg-Trp-Cys]- NH₂; (SEQ ID NO: 40) Ac-Arg-cyclo[Cys-D-Ala-D-Pro-D-Phe-Arg-Trp-Cys]- NH₂; (SEQ ID NO: 2) Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 4) Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe(p-F)-Arg-Trp-  Cys]-NH₂;(SEQ ID NO: 41) Ac-Arg-cyclo[Cys-D-Ala-Atc-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 42) Ac-Arg-cyclo[Cys-D-Ala-QAla-D-Phe-Arg-Trp-Cys]- NH₂; (SEQ ID NO: 43) Ac-Arg-cyclo[Cys-D-Ala-sChp-D-Phe-Arg-Trp-Cys]- NH₂;  or(SEQ ID NO: 44) Ac-Arg-cyclo[Cys-D-Ala-X-D-Phe-Arg-Trp-Cys]-NH₂, 

or a pharmaceutically acceptable salt thereof.
 10. The polypeptide ofclaim 1, represented by any one of the following structural formulas:(SEQ ID NO: 37) Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp- Cys]-NH₂; (SEQ ID NO: 8)Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂;  or (SEQ ID NO: 7)Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-NH₂, 

or a pharmaceutically acceptable salt thereof.
 11. The polypeptide ofclaim 1, wherein A² is an amino acid residue selected from Cys, hCys,Pen, Lys, Orn, Dbu, and Dpr.
 12. The polypeptide of claim 1, wherein A⁴is an amino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser,Thr, Pro, Hyp, Asn, Gln, Trp, Tyr, Lys, Arg, sChp, and residue X. 13.The polypeptide of claim 1, wherein A⁴ is Gln.
 14. The polypeptide ofclaim 1, wherein A⁵ is a D-amino acid.
 15. The polypeptide of claim 1,wherein A⁵ is D-Phe.
 16. The polypeptide of claim 1, wherein A⁸ is anamino acid residue selected from Cys, hCys, Pen, Asp, Glu, Orn, Dbu, andDpr.
 17. An isolated polypeptide of the following structural Formula(I):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is —H, or aC1-C6 acyl; R² is —NR³R⁴ or —OR⁵, wherein R³, R⁴, and R⁵ are eachindependently H or a C1-C6 alkyl; A¹ is an amino acid residue selectedfrom Arg, Lys, Orn, His, Nle, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr,Gln, Asn, Asp, Glu, or TzAla; or A¹ is a moiety selected from anoptionally substituted C1-C12 alkyl, an optionally substituted C6-C18aryl, an optionally substituted C5-C18 heteroaryl, an aralkyl whereinthe aryl portion is an optionally substituted C6-C18 aryl, and the alkylportion is an optionally substituted C1-C12 alkyl, or a heteroaralkyl,wherein the heteroaryl portion is an optionally substituted C5-C18heteroaryl, and the alkyl portion is an optionally substituted C1-C12alkyl; or A¹ is absent; or A² is an amino acid residue selected fromCys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr, and A⁸ is an amino acidresidue selected from Cys, hCys, Pen, Asp, Glu, Orn, Dbu, or Dpr,wherein A² and A⁸ are pairwise selected so as to be able to formcovalent bond between their respective side chains; A³ is an amino acidresidue selected from Ala, Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys,Arg, His, Phe, Gln, Sar, Gly, Asn, Aib, or A³ is absent, or A³ isresidue Y, wherein Y is an amino acid selected from amino acidsrepresented by the following structural formulas

wherein: R¹¹ and R¹², each independently, is H, —CH₃, phenyl, or benzyl;R²¹, R²², R²³, and R²⁴, each independently is H, —CH₃, —CF₃, phenyl,benzyl, F, Cl, Br, I, —OCH₃, or —OH; R³¹, R³², R³³, R³⁴, R⁴¹, R⁴², andR⁴³, each independently is H, —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I,—OCH₃, or —OH; A⁴ is absent or is an amino acid residue selected fromAtc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, an optionallysubstituted His, Trp, Tyr, Lys, Arg, sChp, or residue X, where the X isan amino acid selected from amino acids represented by the followingstructural formulas:

wherein: R⁵¹ and R⁵², each independently, is H, —CH₃, phenyl, or benzyl;R⁶¹, R⁶², R⁶³, and R⁶⁴, each independently is H, —CH₃, —CF₃, phenyl,benzyl, F, Cl, Br, I, —OCH₃, or —OH; R⁷¹, R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², andR⁸³, each independently is H, —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I,—OCH₃, or —OH; A⁵ is an optionally substituted Phe, an optionallysubstituted 1-Nal, or an optionally substituted 2-Nal; A⁶ is Arg; and A⁷is Trp, wherein any amino acid residue is either in L- or inD-configuration, provided that: 1) A³ and A⁴ are not both absent; 2)when A⁴ is an amino acid, A³ is not Aib or Gly; and 3) when A⁴ is Hisand A⁵ is a D-Phe or 2-Nal, A³ is not a D-amino acid or L-Ala; 4) whenA² and A⁸ each is selected from Cys, hCys or Pen, then: (a) when A⁴ isabsent, then A³ is not L-His; (b) when A³ is absent, then A⁴ is notL-His; (c) when A⁴ is His, then A³ is not Glu, Leu, or Lys; and 5) whenA² is Asp, Glu, Lys, or Orn, then A⁸ is not Asp, Glu, Dpr, or Orn. 18.The isolated polypeptide of claim 1, wherein: R² is —NR³R⁴; A¹ is anamino acid residue selected from Arg, Glu, TzAla, C1-C12 alkyl, or anaralkyl wherein the aryl portion is an optionally substituted C6-C18aryl; A² is an amino acid residue selected from Cys, hCys, Pen, Asp,Glu, or Dpr, and A⁸ is an amino acid residue selected from Cys, hCys,Pen, Glu, Orn, Dbu, or Dpr, wherein A² and A⁸ are pairwise selected soas to be able to form covalent bond between their respective sidechains; A³ is an amino acid residue selected from Ala, Tle, Val, Leu,Ile, Pro, Ser, Gln, Sar, Aib, or A³ is absent, and A⁴ is absent or is anamino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr,Pro, Hyp, Asn, Gln, an optionally substituted His, Trp, Tyr, Lys, Arg,or sChp.
 19. The polypeptide of claim 1, wherein R¹ is C1-C6 acyl. 20.The polypeptide of claim 1, wherein A¹ is Arg.
 21. The polypeptide ofclaim 1, wherein A² is an amino acid residue selected from Glu, Cys, andhCys.
 22. The polypeptide of claim 1, wherein A³ is an amino acidresidue selected from Ala, Val, and D-Gln.
 23. The polypeptide of claim1, wherein A⁴ is absent.
 24. The polypeptide of claim 1, wherein A⁸ isan amino acid residue selected from Cys, Dpr, and Pen.
 25. Thepolypeptide of claim 1, wherein the polypeptide is selected from any oneof the following structural formulas: (SEQ ID NO: 15)Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 14)Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 45)Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Pen]-NH₂;  (SEQ ID NO: 26)Ac-Arg-cyclo[Glu-D-Ala-D-Phe-Arg-Trp-Dpr]-NH₂;  (SEQ ID NO: 27)Ac-Arg-cyclo[Glu-Ala-D-Phe-Arg-Trp-Dpr]-NH₂;  (SEQ ID NO: 46)Ac-Arg-cyclo[hCys-Aib-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 47)Ac-Arg-cyclo[hCys-Sar-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 48)Ac-Arg-cyclo[hCys-Val-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 49)Ac-Arg-cyclo[hCys-D-Val-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 50)Ac-Arg-cyclo[hCys-Gln-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 51)Ac-Arg-cyclo[hCys-D-Gln-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 52)Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Pen]-NH₂;  (SEQ ID NO: 53)Ac-Arg-cyclo[D-Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂;  (SEQ ID NO: 17)Ac-Arg-cyclo[Cys-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂;  (SEQ ID NO: 54)Ac-Arg-cyclo[Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂;  (SEQ ID NO: 55)Ac-Arg-cyclo[D-hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂;  (SEQ ID NO: 20)Ac-Arg-cyclo[hCys-Pro-D-Phe-Arg-Trp-Cys]-NH₂;  or (SEQ ID NO: 56)Ac-Arg-cyclo[hCys-D-Pro-D-Phe-Arg-Trp-Cys]-NH₂, 

or a pharmaceutically acceptable salt thereof.
 26. The polypeptide ofclaim 1, wherein the polypeptide is selected from any one of thefollowing structural formulas: (SEQ ID NO: 27)Ac-Arg-cyclo[Glu-Ala-D-Phe-Arg-Trp-Dpr]-NH₂; (SEQ ID NO: 48)Ac-Arg-cyclo[hCys-Val-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 51)Ac-Arg-cyclo[hCys-D-Gln-D-Phe-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 52)Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Pen]-NH₂;

and or a pharmaceutically acceptable salt thereof.
 27. A pharmaceuticalcomposition comprising the polypeptide of claim 1 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.